Soybean variety 01064164

ABSTRACT

The invention relates to the soybean variety designated 01064164. Provided by the invention are the seeds, plants and derivatives of the soybean variety 01064164. Also provided by the invention are tissue cultures of the soybean variety 01064164 and the plants regenerated therefrom. Still further provided by the invention are methods for producing soybean plants by crossing the soybean variety 01064164 with itself or another soybean variety and plants produced by such methods.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of soybeanbreeding. In particular, the invention relates to the novel soybeanvariety 01064164.

Description of Related Art

There are numerous steps in the development of any novel plantgermplasm. Plant breeding begins with the analysis and definition ofproblems and weaknesses of the current germplasm, the establishment ofprogram goals, and the definition of specific breeding objectives. Thenext step is selection of germplasm that possess the traits to meet theprogram goals. The goal is to combine in a single variety an improvedcombination of traits from the parental germplasm. These importanttraits may include higher seed yield, resistance to diseases andinsects, better stems and roots, tolerance to drought and heat, betteragronomic quality, resistance to herbicides, and improvements incompositional traits.

Soybean, Glycine max (L.), is a valuable field crop. Thus, a goal ofplant breeders is to develop stable, high-yielding soybean varietiesthat are agronomically sound. The reasons for this goal are to maximizethe amount of grain produced on the land used and to supply food forboth animals and humans. To accomplish this goal, the soybean breedermust select and develop soybean plants that have the traits that resultin superior varieties.

The oil extracted from soybeans is widely used in food products, such asmargarine, cooking oil, and salad dressings. Soybean oil is composed ofsaturated, monounsaturated, and polyunsaturated fatty acids, with atypical composition of 11% palmitic, 4% stearic, 25% oleic, 50%linoleic, and 9% linolenic fatty acid content (“Economic implications ofmodified soybean traits: summary report,” Iowa Soybean Promotion Board,Soybean Trait Modification Task Force, and American Soybean AssociationSpecial Report 92S, May 1990).

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the soybeanvariety 01064164. The invention also relates to plants produced bygrowing the seed of the soybean variety 01064164, as well as thederivatives of such plants. Further provided are plant parts, includingcells, plant protoplasts, plant cells of a tissue culture from whichsoybean plants can be regenerated, plant calli, plant clumps, and plantcells that are intact in plants or plant parts, such as pollen, flowers,seeds, pods, leaves, stems, and the like.

In a further aspect, the invention provides a composition comprising aseed of soybean variety 01064164 comprised in plant seed growth media.In certain embodiments, the plant seed growth media is a soil orsynthetic cultivation medium. In specific embodiments, the growth mediummay be comprised in a container or may, for example, be soil in a field.Plant seed growth media are well known to those of skill in the art andinclude, but are in no way limited to, soil or synthetic cultivationmedium. Plant seed growth media can provide adequate physical supportfor seeds and can retain moisture and/or nutritional components.Examples of characteristics for soils that may be desirable in certainembodiments can be found, for instance, in U.S. Pat. Nos. 3,932,166 and4,707,176. Synthetic plant cultivation media are also well known in theart and may, in certain embodiments, comprise polymers or hydrogels.Examples of such compositions are described, for example, in U.S. Pat.No. 4,241,537.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the soybean variety 01064164, as well as plantsregenerated therefrom, wherein the regenerated soybean plant is capableof expressing all the morphological and physiological characteristics ofa plant grown from the soybean seed designated 01064164.

Yet another aspect of the current invention is a soybean plant furthercomprising a single locus conversion. In one embodiment, the soybeanplant is defined as further comprising the single locus conversion andotherwise capable of expressing all of the morphological andphysiological characteristics of the soybean variety 01064164. Inparticular embodiments of the invention, the single locus conversion maycomprise a transgenic gene which has been introduced by genetictransformation into the soybean variety 01064164 or a progenitorthereof. In still other embodiments of the invention, the single locusconversion may comprise a dominant or recessive allele. The locusconversion may confer potentially any trait upon the single locusconverted plant, including herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral disease, male fertility orsterility, and improved nutritional quality. In certain embodiments, apotential locus conversion that confers herbicide resistance may conferresistance to herbicides such as, for example, imidazolinone herbicides,sulfonylurea herbicides, triazine herbicides, phenoxy herbicides,cyclohexanedione herbicides, benzonitrile herbicides,4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicides,protoporphyrinogen oxidase-inhibiting herbicides, acetolactatesynthase-inhibiting herbicides, 1-aminocyclopropane-1-carboxylic acidsynthase-inhibiting herbicides, bromoxynil, nicosulfuron,2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, quizalofop-p-ethyl,glyphosate, or glufosinate.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid soybean seed produced by crossing a plant of the soybeanvariety 01064164 to a second soybean plant. Also included in theinvention are the F₁ hybrid soybean plants grown from the hybrid seedproduced by crossing the soybean variety 01064164 to a second soybeanplant.

Still yet another aspect of the invention is a method of producingsoybean seeds comprising crossing a plant of the soybean variety01064164 to any second soybean plant, including itself or another plantof the variety 01064164. In particular embodiments of the invention, themethod of crossing comprises the steps of a) planting seeds of thesoybean variety 01064164; b) cultivating soybean plants resulting fromsaid seeds until said plants bear flowers; c) allowing fertilization ofthe flowers of said plants; and d) harvesting seeds produced from saidplants.

Still yet another aspect of the invention is a method of producinghybrid soybean seeds comprising crossing the soybean variety 01064164 toa second, distinct soybean plant that is nonisogenic to the soybeanvariety 01064164. In particular embodiments of the invention, thecrossing comprises the steps of a) planting seeds of soybean variety01064164 and a second, distinct soybean plant, b) cultivating thesoybean plants grown from the seeds until the plants bear flowers; c)cross-pollinating a flower on one of the two plants with the pollen ofthe other plant, and d) harvesting the seeds resulting from thecross-pollinating.

Still yet another aspect of the invention is a method for developing asoybean plant in a soybean breeding program comprising: obtaining asoybean plant, or its parts, of the variety 01064164; and b) employingsaid plant or parts as a source of breeding material using plantbreeding techniques. In the method, the plant breeding techniques may beselected from the group consisting of recurrent selection, massselection, bulk selection, backcrossing, pedigree breeding, geneticmarker-assisted selection and genetic transformation. In certainembodiments of the invention, the soybean plant of variety 01064164 isused as the male or female parent.

Still yet another aspect of the invention is a method of producing asoybean plant derived from the soybean variety 01064164, the methodcomprising the steps of: (a) crossing a plant of the soybean variety01064164 with a second soybean plant to produce a progeny plant that isderived from soybean variety 01064164; and (b) crossing the progenyplant with itself or a second plant to produce a progeny plant of asubsequent generation that is derived from a plant of the soybeanvariety 01064164. In one embodiment of the invention, the method furthercomprises: (c) crossing the progeny plant of a subsequent generationwith itself or a second plant to produce a progeny plant of a furthersubsequent generation that is derived from a plant of the soybeanvariety 01064164; and (d) repeating step (c), in some embodiments, atleast 2, 3, 4 or more additional generations to produce an inbredsoybean plant that is derived from the soybean variety 01064164. Theinvention still further provides a soybean plant produced by this andthe foregoing methods.

In another embodiment of the invention, the method of producing asoybean plant derived from the soybean variety 01064164 furthercomprises: (a) crossing the soybean variety 01064164-derived soybeanplant with itself or another soybean plant to yield additional soybeanvariety 01064164-derived progeny soybean seed; (b) growing the progenysoybean seed of step (a) under plant growth conditions to yieldadditional soybean variety 01064164-derived soybean plants; and (c)repeating the crossing and growing steps of (a) and (b) to generatefurther soybean variety 01064164-derived soybean plants. In specificembodiments, steps (a) and (b) may be repeated at least 1, 2, 3, 4, or 5or more times as desired. The invention still further provides a soybeanplant produced by this and the foregoing methods.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods and composition relating toplants, seeds and derivatives of the soybean variety 01064164. Soybeanvariety 01064164 is adapted to Mid Group 00. Soybean variety 01064164was developed from an initial cross ofRB00521R2N/(AG00931*2/A3525-A3244-BAH:0001>[0001,0003,0005-0007,0014,0015,0017,0019-0021,0026,0029-0032,0036,0043,0046,0049].@e-0020. @. @.). The breeding history of the variety can be summarizedas follows:

Generation Year Description Cross 2012 The cross was made near Guelph,Ontario. F₁ 2012 Plants were grown near Kunia, HI and advanced usingbulk. F₂ 2012 Plants were grown near Kunia, HI and advanced using singleplant selection. F₃ 2013 Plants were grown near Guelph, Ontario andadvanced using single plant selection. F₄ 2013 Plants were grown nearRancagua, Chile in progeny rows and the variety 01064164 was selectedbased on the agronomic characteristics, general phenotypic appearance,and traits of interest based on molecular marker information. YieldTesting Generation Year No. of Locations Rank No. of Entries F₅ 2014  4 7 50 F₆ 2015 13 10 50 F₇ 2016 21 18 36

The soybean variety 01064164 has been judged to be uniform for breedingpurposes and testing. The variety 01064164 can be reproduced by plantingand growing seeds of the variety under self-pollinating orsib-pollinating conditions, as is known to those of skill in theagricultural arts. Variety 01064164 shows no variants other than whatwould normally be expected due to environment or that would occur foralmost any characteristic during the course of repeated sexualreproduction.

The results of an objective evaluation of the variety are presentedbelow, in Table 1. Those of skill in the art will recognize that theseare typical values that may vary due to environment and that othervalues that are substantially equivalent are within the scope of theinvention. An ‘*’ denotes classifications/scores generated based ongreenhouse assays.

TABLE 1 Phenotypic Description of Variety 01064164 Trait PhenotypeMorphology Relative Maturity 0.04 Flower Color Purple Pubescence ColorTawny Hilum Color Black Pod Color Brown Seed Coat Color Yellow Seed CoatLuster Dull Seed Shape Spherical Flattened Cotyledon Color Yellow LeafShape Ovate Leaf Color Green Canopy Intermediate Growth HabitIndeterminate Disease Reactions Phytophthora Allele* None PhytophthoraTolerance* Moderately Tolerant to Race 25 Soybean Cyst Nematode Race 1*Susceptible Soybean Cyst Nematode Race 3* Susceptible Brown Stem Rot*Highly Susceptible Chloride Sensitivity* Includer Herbicide ReactionsGlyphosate Resistant, MON89788 Sulfonylurea Susceptible DicambaResistant, MON87708 Fatty Acid Fatty Acid Composition Normal

As disclosed herein above, soybean variety 01064164 contains eventsMON89788 and MON87708. Event MON89788, also known as event GM_A19788,confers glyphosate tolerance and is the subject of U.S. Pat. No.7,632,985, the disclosure of which is incorporated herein by reference.Event MON89788 is also covered by one or more of the following patents:U.S. Pat. Nos. 6,051,753; 6,660,911; 6,949,696; 7,141,722; 7,608,761;8,053,184; and 9,017,947. Event MON87708 confers dicamba tolerance andis the subject of U.S. Pat. No. 8,501,407, the disclosure of which isincorporated herein by reference. Event MON87708 is also covered by oneor more of the following patents: U.S. Pat. Nos. 5,850,019; 7,812,224;7,838,729; 7,884,262; 7,939,721; 8,119,380; 8,207,092; 8,629,323;8,754,011; and RE45,048.

The performance characteristics of soybean variety 01064164 were alsoanalyzed and comparisons were made with selected varieties. The resultsof the analysis are presented below, in Table 2.

TABLE 2 Exemplary Agronomic Traits of Variety 01064164 and SelectedVarieties Entries Compared YLD_BE MAT PHT LDG EMERG PRO OIL SWT SDV01064164 42.52 8.63 31.00 2.83 1.57 40.06 21.63 2576.43 AG00632 39.097.30 28.22 2.32 1.42 39.83 21.67 2306.08 Deviation 3.43 1.32 2.78 0.500.15 0.23 −0.04 270.34 Significance ** ** + * * # Obs 35 20 6 13 11 8 85 Years 2 2 2 2 2 2 2 2 Win Percent 77.1 15.0 16.7 25.0 57.1 50.0 62.5100.0 Test Mean 40.84 9.41 27.48 2.47 1.51 39.80 21.67 2664.04 0106416442.52 7.74 31.00 2.83 1.57 40.06 21.63 2576.43 AG0134 43.64 13.45 32.003.64 1.38 38.21 22.39 2779.33 Deviation −1.12 −5.71 −1.00 −0.81 0.191.86 −0.77 −202.91 Significance ** * ** ** + # Obs 35 19 6 13 11 8 8 5Years 2 2 2 2 2 2 2 2 Win Percent 37.1 100.0 50.0 75.0 40.0 87.5 12.520.0 Test Mean 43.04 12.76 29.26 2.43 1.47 39.82 21.58 2733.93 0106416441.64 6.78 28.17 2.65 1.92 40.62 21.60 2390.42 2.73 CX00903 44.96 11.7228.01 2.54 1.82 40.99 20.50 2888.23 3.16 Deviation −3.32 −4.94 0.15 0.100.10 −0.37 1.10 −497.82 −0.43 Significance ** ** * # Obs 38 23 10 20 185 5 2 5 Years 2 2 2 2 2 2 2 1 1 Win Percent 23.7 100.0 60.0 42.9 36.440.0 100.0 0.0 60.0 Test Mean 44.21 12.12 28.11 2.44 1.81 40.42 21.602552.07 2.72 01064164 43.15 8.70 32.49 2.89 1.89 40.06 21.63 2576.433.00 CX00704N 43.63 8.18 27.47 1.82 1.86 39.58 21.54 2709.78 3.08Deviation −0.49 0.52 5.01 1.07 0.02 0.49 0.08 −133.36 −0.08 Significance** ** + # Obs 48 33 17 26 24 8 8 5 11 Years 2 2 2 2 2 2 2 2 1 WinPercent 54.2 36.7 0.0 5.0 46.2 75.0 71.4 40.0 50.0 Test Mean 41.95 8.3930.35 2.49 1.81 39.58 21.72 2635.87 3.02 01064164 43.12 8.70 32.49 3.001.89 40.06 21.63 2576.43 3.00 AG008X7 41.87 8.16 30.97 1.82 1.82 39.1521.77 2679.85 3.27 Deviation 1.25 0.54 1.52 1.19 0.06 0.91 −0.14 −103.42−0.27 Significance * * ** ** * # Obs 49 33 17 27 24 8 8 5 11 Years 2 2 22 2 2 2 2 1 Win Percent 65.3 34.5 23.5 18.2 42.9 87.5 37.5 20.0 87.5Test Mean 42.26 9.07 30.80 2.58 1.81 39.62 21.67 2625.73 3.05 0106416442.27 7.64 31.00 2.83 1.57 40.06 21.63 2576.43 AG005X7 39.32 5.38 26.733.34 1.52 41.18 21.28 2706.61 Deviation 2.94 2.26 4.27 −0.51 0.05 −1.120.34 −130.18 Significance ** ** * ** # Obs 29 18 6 13 11 8 8 5 Years 2 22 2 2 2 2 2 Win Percent 69.0 12.5 16.7 70.0 71.4 12.5 71.4 40.0 TestMean 40.97 6.35 27.79 2.55 1.42 39.94 21.64 2692.22 01064164 42.53 8.1432.25 2.50 1.00 CR00514 38.30 5.31 26.69 1.41 1.01 Deviation 4.24 2.835.56 1.09 −0.01 Significance ** ** * ** # Obs 29 18 4 7 4 Years 2 2 2 11 Win Percent 75.9 5.6 0.0 0.0 100.0 Test Mean 40.30 9.00 28.13 2.231.19 01064164 42.80 8.63 31.00 2.67 1.35 40.06 21.63 2576.43 AG0093240.73 10.96 29.31 2.32 1.27 40.30 20.95 2545.13 Deviation 2.07 −2.331.69 0.35 0.08 −0.24 0.67 31.30 Significance * ** + * ** # Obs 34 20 612 10 8 8 5 Years 2 2 2 2 2 2 2 2 Win Percent 82.4 85.0 16.7 22.2 60.025.0 100.0 60.0 Tcst Mean 42.11 11.23 28.65 2.28 1.26 39.82 21.582646.83 01064164 43.36 8.70 32.49 2.81 1.80 40.06 21.63 2576.43 3.00AG0231 45.47 12.30 32.32 2.40 1.67 39.62 21.19 2600.47 2.81 Deviation−2.10 −3.59 0.17 0.41 0.14 0.45 0.43 −24.04 0.19 Significance *** * + * * # Obs 47 33 17 25 23 8 8 5 11 Years 2 2 2 2 2 2 2 2 1 WinPercent 40.4 93.9 46.7 22.2 20.0 75.0 87.5 40.0 22.2 Test Mean 43.6611.52 31.07 2.49 1.70 39.75 21.61 2709.47 3.06 01064164 42.98 8.63 31.002.67 1.35 40.27 21.57 2518.59 23-10RY 35.45 2.86 23.84 1.56 1.27 40.5821.47 2498.17 Deviation 7.53 5.76 7.16 1.11 0.07 −0.31 0.10 20.41Significance ** ** ** ** # Obs 33 20 6 12 10 7 7 4 Years 2 2 2 2 2 2 2 2Win Percent 90.9 0.0 0.0 10.0 66.7 28.6 57.1 75.0 Test Mean 39.49 7.2026.62 2.35 1.22 40.01 21.69 2664.66 01064164 43.36 8.70 32.49 2.81 1.8040.06 21.63 2576.43 3.00 24-10RY 40.47 6.58 27.36 2.13 1.68 40.32 20.812333.30 3.36 Deviation 2.89 2.13 5.12 0.69 0.12 −0.26 0.81 243.13 −0.36Significance ** ** ** ** + ** + ** # Obs 47 33 17 25 23 8 8 5 11 Years 22 2 2 2 2 2 2 1 Win Percent 70.2 21.2 0.0 17.6 25.0 37.5 100.0 80.0 88.9Test Mean 42.23 8.31 30.76 2.45 1.73 39.80 21.59 2653.90 3.05 0106416442.80 8.63 31.00 2.67 1.35 40.06 21.63 2576.43 AG00835 40.77 6.72 27.601.44 1.25 38.47 22.10 2444.88 Deviation 2.04 1.91 3.40 1.22 0.11 1.59−0.48 131.54 Significance + ** * ** ** + # Obs 34 20 6 12 10 8 8 5 Years2 2 2 2 2 2 2 2 Win Percent 61.8 31.6 0.0 10.0 50.0 100.0 14.3 75.0 TestMean 41.82 8.31 28.55 2.44 1.26 39.73 21.66 2653.90 01064164 42.80 8.6331.00 2.67 1.35 40.06 21.63 2576.43 RB00521R2N 40.08 6.14 28.88 2.491.20 39.94 21.38 2296.56 Deviation 2.72 2.49 2.13 0.18 0.15 0.13 0.24279.87 Significance ** ** * # Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 22 2 Win Percent 70.6 0.0 33.3 54.5 0.0 50.0 75.0 100.0 Test Mean 41.688.24 28.44 2.40 1.26 39.80 21.66 2664.04 01064164 42.60 7.64 31.00 2.671.35 40.06 21.63 2576.43 CR00504 44.02 6.11 26.18 1.32 1.10 39.64 22.292586.41 Deviation −1.42 1.53 4.82 1.35 0.25 0.42 −0.67 −9.98Significance ** ** ** ** # Obs 28 18 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2Win Percent 46.4 13.3 0.0 0.0 25.0 71.4 0.0 60.0 Test Mean 42.21 6.0927.18 2.45 1.22 40.10 21.61 2702.36 01064164 42.80 8.63 31.00 2.67 1.3540.06 21.63 2576.43 CR00702N 39.34 5.83 29.17 3.12 1.26 39.59 21.302307.44 Deviation 3.46 2.80 1.83 −0.45 0.09 0.47 0.33 268.98Significance ** ** + + ** # Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 2 22 Win Percent 85.3 10.0 16.7 54.5 50.0 75.0 75.0 100.0 Test Mean 42.218.76 28.44 2.39 1.25 39.89 21.63 2674.19 01064164 42.80 8.63 31.00 2.671.35 40.06 21.63 2576.43 CR00803N 44.05 14.56 29.74 3.08 1.27 40.2821.74 2699.96 Deviation −1.24 −5.93 1.26 −0.42 0.08 −0.22 −0.12 −123.53Significance ** + # Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2 WinPercent 32.4 100.0 16.7 60.0 40.0 37.5 25.0 60.0 Test Mean 42.80 9.1728.88 2.53 1.25 39.86 21.60 2683.58 01064164 44.98 9.85 34.00 6.50 1.7539.00 21.75 2700.43 CR00714 42.03 8.82 30.17 4.00 1.00 39.26 22.182697.41 Deviation 2.94 1.02 3.83 2.50 0.75 −0.26 −0.43 3.02 Significance** * * ** * # Obs 13 10 3 1 2 4 4 3 Years 1 1 1 1 1 1 1 1 Win Percent100.0 11.1 0.0 0.0 0.0 50.0 0.0 66.7 Test Mean 42.60 10.46 31.11 5.221.39 38.99 21.64 2779.07 01064164 42.80 8.63 31.00 2.67 1.35 40.06 21.632576.43 RB00701R2 44.86 11.45 29.25 1.96 1.30 38.57 22.10 2444.88Deviation −2.06 −2.83 1.75 0.71 0.05 1.50 −0.48 131.54 Significance *** * ** ** # Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2 Win Percent38.2 100.0 33.3 12.5 50.0 100.0 0.0 80.0 Test Mean 42.15 9.40 29.76 2.521.28 39.60 21.65 2625.73 01064164 42.80 8.63 31.00 2.67 1.35 40.06 21.632576.43 CR00814 43.59 10.50 31.75 1.98 1.35 40.03 21.71 2376.84Deviation −0.79 −1.88 −0.75 0.69 0.00 0.04 −0.09 199.58 Significance *** # Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2 Win Percent 38.2 65.080.0 0.0 75.0 50.0 50.0 80.0 Test Mean 42.62 9.37 29.56 2.45 1.28 39.6121.65 2625.73 01064164 44.98 9.85 34.00 6.50 1.75 39.00 21.75 2700.43NSC GLADSTONE RR2Y 40.62 8.10 30.00 4.50 1.75 38.92 21.15 2317.90Deviation 4.36 1.75 4.00 2.00 0.00 0.08 0.60 382.54 Significance ** **** * + # Obs 13 10 3 1 2 4 4 3 Years 1 1 1 1 1 1 1 1 Win Percent 92.310.0 0.0 0.0 −0.0 50.0 100.0 100.0 Test Mean 42.98 11.52 32.04 5.38 1.6138.59 21.74 2732.12 01064164 42.80 7.74 31.00 2.67 1.35 40.06 21.632576.43 CR00804 43.41 7.05 24.88 1.35 1.27 39.09 21.71 2525.62 Deviation−0.61 0.68 6.13 1.31 0.07 0.97 −0.09 50.80 Significance ** ** + # Obs 3419 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2 Win Percent 41.2 33.3 0.0 0.060.0 75.0 50.00 40.0 Test Mean 42.68 8.68 28.12 2.35 1.23 39.81 21.702667.11 01064164 42.80 8.63 31.00 2.67 1.35 40.06 21.63 2576.43 CR011046.99 14.40 33.01 4.01 1.26 38.95 22.80 2982.40 Deviation −4.19 −5.78−2.01 −1.34 0.09 1.11 −1.18 −405.97 Significance ** ** + ** * ** ** #Obs 34 20 6 12 10 8 8 5 Years 2 2 2 2 2 2 2 2 Win Percent 20.6 100.080.0 81.8 75.0 87.5 12.5 0.0 Test Mean 44.73 12.48 30.15 2.38 1.24 39.7621.59 2684.79 01064164 44.98 9.85 34.00 6.50 1.75 39.00 21.75 2700.4324-61RY 42.73 11.10 33.33 6.00 1.75 38.75 22.43 2771.50 Deviation 2.25−1.25 0.67 0.50 0.00 0.25 −0.67 −71.06 Significance * * ** * ** # Obs 1210 3 1 2 4 4 3 Years 1 1 1 1 1 1 1 1 Win Percent 75.0 88.9 33.3 0.0 −0.0100.0 0.0 33.3 Test Mean 42.96 11.52 32.04 5.38 1.61 38.59 21.74 2732.1201064164 41.46 7.40 28.00 2.32 1.25 41.13 21.50 2390.42 AG00937 45.069.15 26.92 1.38 1.11 40.43 21.34 2390.42 Deviation −3.60 −1.75 1.08 0.940.14 0.70 0.16 0.00 Significance * + ** # Obs 21 10 3 11 8 4 4 2 Years 11 1 1 1 1 1 1 Win Percent 28.6 80.0 33.3 12.5 0.0 50.0 66.7 50.0 TestMean 42.16 7.36 26.85 2.19 1.22 40.63 21.59 2466.14 01064164 41.46 7.4028.00 2.32 1.25 41.13 21.50 2390.42 RB00751R2 42.07 6.90 26.17 3.64 1.1040.08 21.68 2549.18 Deviation −0.61 0.50 1.83 −1.32 0.15 1.04 −0.18−158.76 Significance * * # Obs 21 10 3 11 8 4 4 2 Years 1 1 1 1 1 1 1 1Win Percent 47.6 50.0 50.0 77.8 33.3 100.0 25.0 0.0 Test Mean 42.90 7.4927.07 2.26 1.22 40.71 21.51 2466.14 01064164 42.75 8.20 32.16 2.66 1.8141.13 21.50 2390.42 3.00 24-12RY 41.88 6.11 28.96 2.27 1.88 41.57 21.402426.71 3.03 Deviation 0.87 2.09 3.20 0.39 −0.07 −0.45 0.10 −36.29 −0.03Significance ** ** + # Obs 34 23 14 24 21 4 4 2 11 Years 1 1 1 1 1 1 1 11 Win Percent 58.8 13.0 7.1 29.4 63.6 0.0 66.7 50.0 37.5 Test Mean 42.087.38 30.68 2.33 1.76 40.62 21.54 2466.14 3.05 01064164 41.46 7.40 28.002.32 1.25 41.17 21.37 2390.42 CR00904N 40.28 8.20 25.17 3.05 1.19 39.6321.60 3193.27 Deviation 1.18 −0.80 2.83 −0.73 0.06 1.53 −0.23 −802.85Significance * + # Obs 21 10 3 11 8 3 3 2 Years 1 1 1 1 1 1 1 1 WinPercent 61.9 70.0 0.0 62.5 50.0 100.0 33.3 0.0 Test Mean 41.63 7.2827.47 2.27 1.20 40.57 21.41 2466.14 **, *, + Significant at P ≤ 0.01,0.05, or 0.10, respectively.

Breeding Soybean Variety 01064164

One aspect of the current invention concerns methods for crossing thesoybean variety 01064164 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of the soybean variety 01064164, or can be used to producehybrid soybean seeds and the plants grown therefrom. Hybrid soybeanplants can be used by farmers in the commercial production of soyproducts or may be advanced in certain breeding protocols for theproduction of novel soybean varieties. A hybrid plant can also be usedas a recurrent parent at any given stage in a backcrossing protocolduring the production of a single locus conversion of the soybeanvariety 01064164.

Soybean variety 01064164 is well suited to the development of newvarieties based on the elite nature of the genetic background of thevariety. In selecting a second plant to cross with 01064164 for thepurpose of developing novel soybean varieties, it will typically bedesired to choose those plants that either themselves exhibit one ormore selected characteristics or that exhibit the characteristic(s) whenin hybrid combination. Examples of potentially selected characteristicsinclude seed yield, lodging resistance, emergence, seedling vigor,disease tolerance, maturity, plant height, high oil content, highprotein content and shattering resistance.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F₁ hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective;whereas, for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcrossing.

The complexity of inheritance influences the choice of the breedingmethod. Backcross breeding is used to transfer one or a few genes for ahighly heritable trait into a desirable variety. This approach has beenused extensively for breeding disease-resistant varieties (Bowers etal., Crop Sci., 32(1):67-72, 1992; Nickell and Bernard, Crop Sci.,32(3):835, 1992). Various recurrent selection techniques are used toimprove quantitatively inherited traits controlled by numerous genes.The use of recurrent selection in self-pollinating crops depends on theease of pollination, the frequency of successful hybrids from eachpollination, and the number of hybrid offspring from each successfulcross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulvarieties produced per unit of input, e.g., per year, per dollarexpended, etc.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments that are representative of thecommercial target area(s) for generally three or more years. The bestlines are candidates for new commercial varieties. Those still deficientin a few traits may be used as parents to produce new populations forfurther selection.

These processes, which lead to the final step of marketing anddistribution, may take as much as eight to 12 years from the time thefirst cross is made. Therefore, development of new varieties is atime-consuming process that requires precise forward planning, efficientresource utilization, and minimal direction changes.

Identifying individuals that are genetically superior is a difficulttask because the true genotypic value for most traits can be masked byother confounding traits or environmental factors. One method ofidentifying a superior plant is observing its performance relative toother experimental plants and one or more widely grown standardvarieties. Single observations are generally inconclusive, whilereplicated observations provide a better estimate of genetic worth.

The goal of plant breeding is to develop new, unique, and superiorsoybean varieties and hybrids. The breeder initially selects and crossestwo or more parental lines. This is generally followed by repeatedselfing and selection, which produces many new genetic combinations.Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic, and soil conditions, and further selections arethen made during and at the end of the growing season. The varietieswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a gross and general fashion. Thesame breeder cannot produce the same variety twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new soybean varieties.

Pedigree breeding and recurrent selection breeding methods are used todevelop varieties from breeding populations. Breeding programs combinetraits from two or more varieties or various broad-based sources intobreeding pools from which varieties are developed by selfing andselection of phenotypes. The new varieties are evaluated to determinewhich have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce F₁ progeny. An F₂ populationis then produced by selfing one or several F₁ plants. Selection of thebest individuals may begin in the F₂ population or later depending uponthe breeder's objectives; then, beginning in the F₃ generation, the bestindividuals in the best families can be selected. Replicated testing offamilies can begin in the F₃ or F₄ generations to improve theeffectiveness of selection for traits of low heritability. At anadvanced stage of inbreeding (i.e., the F₆ and F₇ generations), the bestlines or mixtures of phenotypically similar lines are tested forpotential release as new varieties.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation from which further cycles of selection are continued.

Backcross breeding has been used to transfer genetic loci for simplyinherited or highly heritable traits into a homozygous variety that isused as the recurrent parent. The source of the trait to be transferredis called the donor or nonrecurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent and the traittransferred from the donor parent. After the initial cross, individualspossessing the phenotype of the donor parent are selected and repeatedlycrossed, i.e., backcrossed, to the recurrent parent. The resulting plantis expected to have the attributes of the recurrent parent (i.e.,variety) and the desirable trait transferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which the lines are derived will each traceto different F₂ individuals. The number of plants in a populationdeclines each generation due to failure of some seeds to germinate orsome plants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. This procedure is also referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand as is required for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, “Principles of Plant Breeding,” John Wiley & Sons,NY, University of California, Davis, Calif., 50-98, 1960; Simmonds,“Principles of Crop Improvement,” Longman, Inc., NY, 369-399, 1979;Sneep et al., “Plant breeding perspectives,” Wageningen (ed), Centre forAgricultural Publishing and Documentation, 1979; Fehr, In: “Soybeans:Improvement, Production and Uses,” 2d Ed., Monograph 16:249, 1987; Fehr,“Principles of Cultivar Development,” Theory and Technique (Vol 1) andCrop Species Soybean (Vol 2), Iowa State Univ., Macmillian Pub. Co., NY,360-376, 1987; Poehlman and Sleper, “Breeding Field Crops”, 4th Ed.,Iowa State University Press, Ames, 1995; Sprague and Dudley, eds., Cornand Improvement, 5th ed., 2006).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, there must be a demand for a new varietythat is compatible with industry standards or which creates a newmarket. The introduction of a new variety will incur additional costs tothe seed producer, the grower, processor, and consumer due in part tospecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new variety should take into consideration research anddevelopment costs as well as the technical superiority of the finalvariety. For seed-propagated varieties, it must be feasible to produceseed easily and economically.

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through amolecular marker profile, which can identify plants of the same varietyor a related variety, can identify plants and plant parts which aregenetically superior as a result of an event comprising a backcrossconversion, transgene, or genetic sterility factor, or can be used todetermine or validate a pedigree. Such molecular marker profiling can beaccomplished using a variety of techniques including, but not limitedto, restriction fragment length polymorphism (RFLP), amplified fragmentlength polymorphism (AFLP), sequence-tagged sites (STS), randomlyamplified polymorphic DNA (RAPD), arbitrarily primed polymerase chainreaction (AP-PCR), DNA amplification fingerprinting (DAF), sequencecharacterized amplified regions (SCARs), variable number tandem repeat(VNTR), short tandem repeat (STR), single feature polymorphism (SFP),simple sequence length polymorphism (SSLP), restriction site associatedDNA, allozymes, isozyme markers, single nucleotide polymorphisms (SNPs),or simple sequence repeat (SSR) markers, also known as microsatellites(Gupta et al., 1999; Korzun et al., 2001). Various types of thesemarkers, for example, can be used to identify individual varietiesdeveloped from specific parent varieties, as well as cells or otherplant parts thereof. For example, see Cregan et al. (1999) “AnIntegrated Genetic Linkage Map of the Soybean Genome” Crop Science39:1464-1490, and Berry et al. (2003) “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize InbredLines and Soybean Varieties” Genetics 165(1):331-342, each of which areincorporated by reference herein in their entirety.

In some examples, one or more markers may be used to characterize and/orevaluate a soybean variety. Particular markers used for these purposesare not limited to any particular set of markers, but are envisioned toinclude any type of marker and marker profile that provides a means fordistinguishing varieties. One method of comparison may be to use onlyhomozygous loci for soybean variety 01064164.

Primers and PCR protocols for assaying these and other markers aredisclosed in, for example, Soybase (sponsored by the USDA AgriculturalResearch Service and Iowa State University) located on the world wideweb at 129.186.26/94/SSR.html. In addition to being used foridentification of soybean variety 01064164, as well as plant parts andplant cells of soybean variety 01064164, a genetic profile may be usedto identify a soybean plant produced through the use of soybean variety01064164 or to verify a pedigree for progeny plants produced through theuse of soybean variety 01064164. A genetic marker profile may also beuseful in breeding and developing backcross conversions.

In an embodiment, the present invention provides a soybean plantcharacterized by molecular and physiological data obtained from arepresentative sample of said variety deposited with the American TypeCulture Collection (ATCC). Thus, plants, seeds, or parts thereof, havingall or essentially all of the morphological and physiologicalcharacteristics of soybean variety 01064164 are provided. Furtherprovided is a soybean plant formed by the combination of the disclosedsoybean plant or plant cell with another soybean plant or cell andcomprising the homozygous alleles of the variety.

In some examples, a plant, a plant part, or a seed of soybean variety01064164 may be characterized by producing a molecular profile. Amolecular profile may include, but is not limited to, one or moregenotypic and/or phenotypic profile(s). A genotypic profile may include,but is not limited to, a marker profile, such as a genetic map, alinkage map, a trait maker profile, a SNP profile, an SSR profile, agenome-wide marker profile, a haplotype, and the like. A molecularprofile may also be a nucleic acid sequence profile, and/or a physicalmap. A phenotypic profile may include, but is not limited to, a proteinexpression profile, a metabolic profile, an mRNA expression profile, andthe like.

One means of performing genetic marker profiles is using SSRpolymorphisms that are well known in the art. A marker system based onSSRs can be highly informative in linkage analysis relative to othermarker systems, in that multiple alleles may be present. Anotheradvantage of this type of marker is that through use of flankingprimers, detection of SSRs can be achieved, for example, by using thepolymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. PCR detection may be performedusing two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA to amplify the SSR region.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which correlates to the number of base pairsof the fragment. While variation in the primer used or in the laboratoryprocedures can affect the reported fragment size, relative values shouldremain constant regardless of specific primer or laboratory used. Whencomparing varieties, it may be beneficial to have all profiles performedin the same lab. Primers that can be used are publically available andmay be found in, for example, Soybase or Cregan et al. (Crop Science39:1464-1490, 1999).

A genotypic profile of soybean variety 01064164 can be used to identifya plant comprising variety 01064164 as a parent, since such plants willcomprise the same homozygous alleles as variety 01064164. Because thesoybean variety is essentially homozygous at all relevant loci, mostloci should have only one type of allele present. In contrast, a geneticmarker profile of an F₁ progeny should be the sum of those parents,e.g., if one parent was homozygous for allele X at a particular locus,and the other parent homozygous for allele Y at that locus, then the F₁progeny will be XY (heterozygous) at that locus. Subsequent generationsof progeny produced by selection and breeding are expected to be ofgenotype XX (homozygous), YY (homozygous), or XY (heterozygous) for thatlocus position. When the F₁ plant is selfed or sibbed for successivefilial generations, the locus should be either X or Y for that position.

In addition, plants and plant parts substantially benefiting from theuse of variety 01064164 in their development, such as variety 01064164comprising a backcross conversion, transgene, or genetic sterilityfactor, may be identified by having a molecular marker profile with ahigh percent identity to soybean variety 01064164. Such a percentidentity might be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or 99.9% identical to soybean variety 01064164.

A genotypic profile of variety 01064164 also can be used to identifyessentially derived varieties and other progeny varieties developed fromthe use of variety 01064164, as well as cells and other plant partsthereof. Plants of the invention include any plant having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of themarkers in the genotypic profile, and that retain 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the morphological andphysiological characteristics of variety 01064164 when grown under thesame conditions. Such plants may be developed using markers well knownin the art. Progeny plants and plant parts produced using variety01064164 may be identified, for example, by having a molecular markerprofile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% geneticcontribution from soybean variety 01064164, as measured by eitherpercent identity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of variety 01064164,such as within 1, 2, 3, 4, or 5 or less cross pollinations to a soybeanplant other than variety 01064164, or a plant that has variety 01064164as a progenitor. Unique molecular profiles may be identified with othermolecular tools, such as SNPs and RFLPs.

Any time the soybean variety 01064164 is crossed with another,different, variety, first generation (F₁) soybean progeny are produced.The hybrid progeny are produced regardless of characteristics of the twovarieties produced. As such, an F₁ hybrid soybean plant may be producedby crossing 01064164 with any second soybean plant. The second soybeanplant may be genetically homogeneous (e.g., inbred) or may itself be ahybrid. Therefore, any F₁ hybrid soybean plant produced by crossingsoybean variety 01064164 with a second soybean plant is a part of thepresent invention.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques (see, e.g., Fehr, “Soybean,” In: Hybridization ofCrop Plants, Fehr and Hadley (eds), Am. Soc. Agron. and Crop Sci. Soc.Am., Madison, Wis., 590-599, 1980). Natural pollination occurs insoybeans either by self-pollination or natural cross-pollination, whichtypically is aided by pollinating organisms. In either natural orartificial crosses, flowering and flowering time are an importantconsideration. Soybean is a short-day plant, but there is considerablegenetic variation for sensitivity to photoperiod (Hamner, “Glycinemax(L.) Merrill,” In: The Induction of Flowering: Some Case Histories,Evans (ed), Cornell Univ. Press, Ithaca, N.Y., 62-89, 1969; Criswell andHume, Crop Sci., 12:657-660, 1972). The critical day length forflowering ranges from about 13 h for genotypes adapted to tropicallatitudes to 24 h for photoperiod-insensitive genotypes grown at higherlatitudes (Shibles et al., “Soybean,” In: Crop Physiology: Some CaseHistories, Evans (ed), Cambridge Univ. Press, Cambridge, England,51-189, 1975). Soybeans seem to be insensitive to day length for 9 daysafter emergence. Photoperiods shorter than the critical day length arerequired for 7 to 26 days to complete flower induction (Borthwick andParker, Bot. Gaz., 100:374-387, 1938; Shanmugasundaram and Tsou, CropSci., 18:598-601, 1978).

Sensitivity to day length is an important consideration when genotypesare grown outside of their area of adaptation. When genotypes adapted totropical latitudes are grown in the field at higher latitudes, they maynot mature before frost occurs. Plants can be induced to flower andmature earlier by creating artificially short days or by grafting (Fehr,“Soybean,” In: Hybridization of Crop Plants, Fehr and Hadley (eds), Am.Soc. Agron. and Crop Sci. Soc. Am., Madison, Wis., 590-599, 1980).Soybeans frequently are grown in winter nurseries located at sea levelin tropical latitudes where day lengths are much shorter than theircritical photoperiod. The short day lengths and warm temperaturesencourage early flowering and seed maturation, and genotypes can producea seed crop in 90 days or fewer after planting. Early flowering isuseful for generation advance when only a few self-pollinated seeds perplant are needed, but not for artificial hybridization because theflowers self-pollinate before they are large enough to manipulate forhybridization. Artificial lighting can be used to extend the natural daylength to about 14.5 h to obtain flowers suitable for hybridization andto increase yields of self-pollinated seed.

The effect of a short photoperiod on flowering and seed yield can bepartly offset by altitude, probably due to the effects of cooltemperature (Major et al., Crop Sci., 15:174-179, 1975). At tropicallatitudes, varieties adapted to the northern U.S. perform more likethose adapted to the southern U.S. at high altitudes than they do at sealevel.

The light level required to delay flowering is dependent on the qualityof light emitted from the source and the genotype being grown. Bluelight with a wavelength of about 480 nm requires more than 30 times theenergy to inhibit flowering as red light with a wavelength of about 640nm (Parker et al., Bot. Gaz., 108:1-26, 1946).

Temperature can also play a significant role in the flowering anddevelopment of soybean plants (Major et al., Crop Sci., 15:174-179,1975). It can influence the time of flowering and suitability of flowersfor hybridization. Temperatures below 21° C. or above 32° C. can reducefloral initiation or seed set (Hamner, “Glycine max(L.) Merrill,” In:The Induction of Flowering: Some Case Histories, Evans (ed), CornellUniv. Press, Ithaca, N.Y., 62-89, 1969; van Schaik and Probst, Agron.J., 50:192-197, 1958). Artificial hybridization is most successfulbetween 26° C. and 32° C. because cooler temperatures reduce pollen shedand result in flowers that self-pollinate before they are large enoughto manipulate. Warmer temperatures frequently are associated withincreased flower abortion caused by moisture stress; however, successfulcrosses are possible at about 35° C. if soil moisture is adequate.

Soybeans have been classified as indeterminate, semi-determinate, anddeterminate based on the abruptness of stem termination after floweringbegins (Bernard and Weiss, “Qualitative genetics,” In: Soybeans:Improvement, Production, and Uses, Caldwell (ed), Am. Soc. of Agron.,Madison, Wis., 117-154, 1973). When grown at their latitude ofadaptation, indeterminate genotypes flower when about one-half of thenodes on the main stem have developed. They have short racemes with fewflowers, and their terminal node has only a few flowers.Semi-determinate genotypes also flower when about one-half of the nodeson the main stem have developed, but node development and flowering onthe main stem stops more abruptly than on indeterminate genotypes. Theirracemes are short and have few flowers, except for the terminal one,which may have several times more flowers than those lower on the plant.Determinate varieties begin flowering when all or most of the nodes onthe main stem have developed. They usually have elongated racemes thatmay be several centimeters in length and may have a large number offlowers. Stem termination and flowering habit are reported to becontrolled by two major genes (Bernard and Weiss, “Qualitativegenetics,” In: Soybeans: Improvement, Production, and Uses, Caldwell(ed), Am. Soc. of Agron., Madison, Wis., 117-154, 1973).

Soybean flowers typically are self-pollinated on the day the corollaopens. The amount of natural crossing, which is typically associatedwith insect vectors such as honeybees, is approximately 1% for adjacentplants within a row and 0.5% between plants in adjacent rows (Boerma andMoradshahi, Crop Sci., 15:858-861, 1975). The structure of soybeanflowers is similar to that of other legume species and consists of acalyx with five sepals, a corolla with five petals, 10 stamens, and apistil (Carlson, “Morphology”, In: Soybeans: Improvement, Production,and Uses, Caldwell (ed), Am. Soc. of Agron., Madison, Wis., 17-95,1973). The calyx encloses the corolla until the day before anthesis. Thecorolla emerges and unfolds to expose a standard, two wing petals, andtwo keel petals. An open flower is about 7 mm long from the base of thecalyx to the tip of the standard and 6 mm wide across the standard. Thepistil consists of a single ovary that contains one to five ovules, astyle that curves toward the standard, and a club-shaped stigma. Thestigma is receptive to pollen about 1 day before anthesis and remainsreceptive for 2 days after anthesis, if the flower petals are notremoved. Filaments of nine stamens are fused, and the one nearest thestandard is free. The stamens form a ring below the stigma until about 1day before anthesis, then their filaments begin to elongate rapidly andelevate the anthers around the stigma. The anthers dehisce on the day ofanthesis, pollen grains fall on the stigma, and within 10 h the pollentubes reach the ovary and fertilization is completed (Johnson andBernard, “Soybean genetics and breeding,” In: The Soybean, Norman (ed),Academic Press, NY, 1-73, 1963).

Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is often beneficial,although not required, to utilize artificial hybridization. Inartificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self-fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

For artificial hybridization employing emasculation, flowers that areexpected to open the following day are selected on the female parent.The buds are swollen and the corolla is just visible through the calyxor has begun to emerge. The selected buds on a parent plant are preparedand all of the self-pollinated flowers or immature buds are removed.Special care is required to remove immature buds that are hidden underthe stipules at the leaf axil, which could develop into flowers at alater date. To remove a flower, the flower is grasped and the locationof the stigma is determined by examining the sepals. A long, curvy sepalcovers the keel, and the stigma is on the opposite side of the flower.The calyx is removed by pulling each sepal down and around the flower.The exposed corolla is then removed just above the calyx scar, takingcare to remove the keel petals without injuring the stigma. The ring ofanthers is visible after the corolla is removed, unless the anthers wereremoved with the petals. Cross-pollination can then be carried outusing, for example, petri dishes or envelopes in which male flowers havebeen collected. Desiccators containing calcium chloride crystals areused in some environments to dry male flowers to obtain adequate pollenshed.

It has been demonstrated that emasculation is unnecessary to preventself-pollination (Walker et al., Crop Sci., 19:285-286, 1979). Whenemasculation is not used, the anthers near the stigma frequently areremoved to make it clearly visible for pollination. The female flowerusually is hand-pollinated immediately after it is prepared; although adelay of several hours does not seem to reduce seed set. Pollen shedtypically begins in the morning and may end when temperatures are above30° C., or may begin later and continue throughout much of the day withmore moderate temperatures.

Pollen is available from a flower with a recently opened corolla, butthe degree of corolla opening associated with pollen shed may varyduring the day. In many environments, it is possible to collect maleflowers and use them immediately without storage. In the southern U.S.and other humid climates, pollen shed occurs in the morning when femaleflowers are more immature and difficult to manipulate than in theafternoon, and the flowers may be damp from heavy dew. In thosecircumstances, male flowers may be collected into envelopes or petridishes in the morning and the open container placed in a desiccator forabout 4 h at a temperature of about 25° C. The desiccator may be takento the field in the afternoon and kept in the shade to prevent excessivetemperatures from developing within it. Pollen viability can bemaintained in flowers for up to 2 days when stored at about 5° C. In adesiccator at 3° C., flowers can be stored successfully for severalweeks; however, varieties may differ in the percentage of pollen thatgerminates after long-term storage (Kuehl, “Pollen viability and stigmareceptivity of Glycine max (L.) Merrill,” Thesis, North Carolina StateCollege, Raleigh, N.C., 1961).

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable orthe same male with good pollen shed may be used to pollinate severalflowers.

When male flowers do not have to be collected and dried in a desiccator,it may be desired to plant the parents of a cross adjacent to eachother. Plants usually are grown in rows 65 to 100 cm apart to facilitatemovement of personnel within the field nursery. Yield of self-pollinatedseed from an individual plant may range from a few seeds to more than1,000 as a function of plant density. A density of 30 plants/m of rowcan be used when 30 or fewer seeds per plant is adequate, 10 plants/mcan be used to obtain about 100 seeds/plant, and 3 plants/m usuallyresults in maximum seed production per plant. Densities of 12 plants/mor less commonly are used for artificial hybridization.

Multiple planting dates about 7 to 14 days apart usually are used tomatch parents of different flowering dates. When differences inflowering dates are extreme between parents, flowering of the laterparent can be hastened by creating an artificially short day orflowering of the earlier parent can be delayed by use of artificiallylong days or delayed planting. For example, crosses with genotypesadapted to the southern U.S. are made in northern U.S. locations bycovering the late genotype with a box, large can, or similar containerto create an artificially short photoperiod of about 12 h for about 15days beginning when there are three nodes with trifoliate leaves on themain stem. Plants induced to flower early tend to have flowers thatself-pollinate when they are small and can be difficult to prepare forhybridization.

Grafting can be used to hasten the flowering of late floweringgenotypes. A scion from a late genotype grafted on a stock that hasbegun to flower will begin to bloom up to 42 days earlier than normal(Kiihl et al., Crop Sci., 17:181-182, 1977). First flowers on the scionappear from 21 to 50 days after the graft.

Observing pod development 7 days after pollination generally is adequateto identify a successful cross. Abortion of pods and seeds can occurseveral weeks after pollination, but the percentage of abortion usuallyis low if plant stress is minimized (Shibles et al., “Soybean,” In: CropPhysiology: Some Case Histories, Evans (ed), Cambridge Univ. Press,Cambridge, England, 51-189, 1975). Pods that develop from artificialhybridization can be distinguished from self-pollinated pods by thepresence of the calyx scar, caused by removal of the sepals. The sepalsbegin to fall off as the pods mature; therefore, harvest should becompleted at or immediately before the time the pods reach their maturecolor. Harvesting pods early also avoids any loss by shattering.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

Further Embodiments of the Invention

In certain aspects of the invention, plants of soybean variety 01064164are modified to include at least a first heritable trait. Such plantsmay, in one embodiment, be developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. By essentially all of the morphological and physiologicalcharacteristics, it is meant that the characteristics of a plant arerecovered that are otherwise present when compared in the sameenvironment, other than occasional variant traits that might ariseduring backcrossing or direct introduction of a transgene. It isunderstood that a locus introduced by backcrossing may or may not betransgenic in origin, and thus the term backcrossing specificallyincludes backcrossing to introduce loci that were created by genetictransformation.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a soybean plant isobtained wherein essentially all of the morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the transferred locus from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the originalvariety. To accomplish this, a locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the genome of theoriginal variety, and therefore the morphological and physiologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially or agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance, itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Soybean varieties can also be developed from more than two parents(Fehr, In: “Soybeans: Improvement, Production and Uses,” 2nd Ed.,Manograph 16:249, 1987). The technique, known as modified backcrossing,uses different recurrent parents during the backcrossing. Modifiedbackcrossing may be used to replace the original recurrent parent with avariety having certain more desirable characteristics or multipleparents may be used to obtain different desirable characteristics fromeach.

Many traits have been identified that are not regularly selected for inthe development of a new inbred but that can be improved by backcrossingtechniques. Traits may or may not be transgenic; examples of thesetraits include, but are not limited to, male sterility, herbicideresistance, resistance to bacterial, fungal, or viral disease, insectand pest resistance, restoration of male fertility, enhanced nutritionalquality, yield stability, and yield enhancement. These comprise genesgenerally inherited through the nucleus.

Direct selection may be applied when the locus acts as a dominant trait.An example of a dominant trait is the herbicide resistance trait. Forthis selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants that have the herbicide resistancegene are used in the subsequent backcross. This process is then repeatedfor all additional backcross generations.

Selection of soybean plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one may utilize a suitable genetic markerthat is closely associated with a trait of interest. One of thesemarkers may therefore be used to identify the presence or absence of atrait in the offspring of a particular cross, and hence may be used inselection of progeny for continued breeding. This technique may commonlybe referred to as marker assisted selection. Any other type of geneticmarker or other assay that is able to identify the relative presence orabsence of a trait of interest in a plant may also be useful forbreeding purposes. Procedures for marker assisted selection applicableto the breeding of soybeans are well known in the art. Such methods willbe of particular utility in the case of recessive traits and variablephenotypes, or when conventional assays may be more expensive, timeconsuming or otherwise disadvantageous. Genetic markers that could beused in accordance with the invention include, but are not necessarilylimited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., Nucleic Acids Res., 18:6531-6535, 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (European Patent Application Publication No.EP0534858, specifically incorporated herein by reference in itsentirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al.,Science, 280:1077-1082, 1998).

Many qualitative characters also have a potential use as phenotype-basedgenetic markers in soybeans; however, some or many may not differ amongvarieties commonly used as parents (Bernard and Weiss, “Qualitativegenetics,” In: Soybeans: Improvement, Production, and Uses, Caldwell(ed), Am. Soc. of Agron., Madison, Wis., 117-154, 1973). The most widelyused genetic markers are flower color (purple dominant to white),pubescence color (brown dominant to gray), and pod color (brown dominantto tan). The association of purple hypocotyl color with purple flowersand green hypocotyl color with white flowers is commonly used toidentify hybrids in the seedling stage. Differences in maturity, height,hilum color, and pest resistance between parents can also be used toverify hybrid plants.

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those that are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the soybean variety of the inventionor may, alternatively, be used for the preparation of transgenes whichcan be introduced by backcrossing. Methods for the transformation ofmany economically important plants, including soybeans, are well knownto those of skill in the art. Techniques which may be employed togenetically transform soybeans include, but are not limited to,electroporation, microprojectile bombardment, Agrobacterium-mediatedtransformation and direct DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Protoplasts may also be employed for electroporation transformation ofplants (Bates, Mol. Biotechnol., 2(2):135-145, 1994; Lazzeri, MethodsMol. Biol., 49:95-106, 1995). For example, the generation of transgenicsoybean plants by electroporation of cotyledon-derived protoplasts wasdescribed by Dhir and Widholm in International Patent ApplicationPublication No. WO 92/17598, the disclosure of which is specificallyincorporated herein by reference.

An efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, or gold. For the bombardment, cells in suspension areconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target soybean cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of theprojectile aggregate and may contribute to a higher frequency oftransformation by reducing the damage inflicted on the recipient cellsby projectiles that are too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. The application ofmicroprojectile bombardment for the transformation of soybeans isdescribed, for example, in U.S. Pat. No. 5,322,783, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio. Tech., 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and cloning sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. Vectors can have convenientmultiple-cloning sites (MCS) flanked by a promoter and a polyadenylationsite for direct expression of inserted polypeptide coding genes. Othervectors can comprise site-specific recombination sequences, enablinginsertion of a desired DNA sequence without the use of restrictionenzymes (Curtis et al., Plant Physiology 133:462-469, 2003).Additionally, Agrobacterium containing both armed and disarmed Ti genescan be used for transformation.

In those plant strains in which Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio. Tech., 3(7):629-635, 1985; U.S.Pat. No. 5,563,055). Use of Agrobacterium in the context of soybeantransformation has been described, for example, by Chee and Slightom(Methods Mol. Biol., 44:101-119, 1995) and in U.S. Pat. No. 5,569,834,the disclosures of which are specifically incorporated herein byreference in their entirety.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199(2):169-177, 1985; Omirulleh etal., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,319(6056):791-793, 1986; Uchimiya et al., Mol. Gen. Genet.,204(2):204-207, 1986; Marcotte et al., Nature, 335(6189):454-457, 1988).The demonstrated ability to regenerate soybean plants from protoplastsmakes each of these techniques applicable to soybean (Dhir et al., PlantCell Rep., 10(2):97-101, 1991).

Included among various plant transformation techniques are methodspermitting the site-specific modification of a plant genome. Thesemodifications can include, but are not limited to, site-specificmutations, deletions, insertions, and replacements of nucleotides. Thesemodifications can be made anywhere within the genome of a plant, forexample, in genomic elements, including, among others, coding sequences,regulatory elements, and non-coding DNA sequences. Any number of suchmodifications can be made and that number of modifications may be madein any order or combination, for example, simultaneously all together orone after another. Such methods may lead to changes in phenotype. Thetechniques for such modifications are well known in the art and include,for example, use of CRISPR-Cas systems, zinc-finger nucleases (ZFNs),and transcription activator-like effector nucleases (TALENs), amongothers.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a soybean plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a soybean plant arepresented below.

A. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. A non-limiting example is a gene conferring resistance toa herbicide that inhibits the growing point or meristem such asimidazolinone or sulfonylurea herbicides. As imidazolinone andsulfonylurea herbicides are acetolactate synthase (ALS)-inhibitingherbicides that prevent the formation of branched chain amino acids,exemplary genes in this category code for ALS and AHAS enzymes asdescribed, for example, by Lee et al., EMBO J., 7:1241, 1988; Gleen etal., Plant Molec. Biology, 18:1185, 1992; and Miki et al., Theor. Appl.Genet., 80:449, 1990. As a non-limiting example, a gene may be employedto confer resistance to the exemplary sulfonylurea herbicidenicosulfuron.

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicusphosphinothricin acetyltransferase (bar) genes) may also be used. See,for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses thenucleotide sequence of a form of EPSPS that can confer glyphosateresistance. Non-limiting examples of EPSPS transformation eventsconferring glyphosate resistance are provided by U.S. Pat. Nos.6,040,497 and 7,632,985. The MON89788 event disclosed in U.S. Pat. No.7,632,985 in particular is beneficial in conferring glyphosate tolerancein combination with an increase in average yield relative to priorevents.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCAccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061 to Comai. A hygromycin Bphosphotransferase gene from E. coli that confers resistance toglyphosate in tobacco callus and plants is described in Penaloza-Vazquezet al., Plant Cell Reports, 14:482, 1995. European Patent ApplicationPublication No. EP0333033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes that confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin acetyltransferase gene isprovided in European Patent Application Publication No. EP0242246 toLeemans et al. DeGreef et al. (Biotechnology, 7:61, 1989) describe theproduction of transgenic plants that express chimeric bar genes codingfor phosphinothricin acetyl transferase activity. Exemplary genesconferring resistance to a phenoxy class herbicide haloxyfop and acyclohexanedione class herbicide sethoxydim are the Acct-S1, Acct-S2 andAcct-S3 genes described by Marshall et al., (Theor. Appl. Genet.,83:435, 1992). As a non-limiting example, a gene may confer resistanceto other exemplary phenoxy class herbicides that include, but are notlimited to, quizalofop-p-ethyl and 2,4-dichlorophenoxyacetic acid(2,4-D).

Genes are also known that confer resistance to herbicides that inhibitphotosynthesis such as, for example, triazine herbicides (psbA and gs+genes) and benzonitrile herbicides (nitrilase gene). As a non-limitingexample, a gene may confer resistance to the exemplary benzonitrileherbicide bromoxynil. Przibila et al. (Plant Cell, 3:169, 1991) describethe transformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (Biochem. J., 285:173, 1992).4-hydroxyphenylpyruvate dioxygenase (HPPD) is a target of theHPPD-inhibiting herbicides, which deplete plant plastoquinone andvitamin E pools. Rippert et al. (Plant Physiol., 134:92, 2004) describesan HPPD-inhibitor resistant tobacco plant that was transformed with ayeast-derived prephenate dehydrogenase (PDH) gene. Protoporphyrinogenoxidase (PPO) is the target of the PPO-inhibitor class of herbicides; aPPO-inhibitor resistant PPO gene was recently identified in Amaranthustuberculatus (Patzoldt et al., PNAS, 103(33):12329, 2006). The herbicidemethyl viologen inhibits CO₂ assimilation. Foyer et al. (Plant Physiol.,109:1047, 1995) describe a plant overexpressing glutathione reductase(GR) that is resistant to methyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides. Modified bacterial genes have beensuccessfully demonstrated to confer resistance to atrazine, a herbicidethat binds to the plastoquinone-binding membrane protein Q_(B) inphotosystem II to inhibit electron transport. See, for example, studiesby Cheung et al. (PNAS, 85:391, 1988), describing tobacco plantsexpressing the chloroplast psbA gene from an atrazine-resistant biotypeof Amaranthus hybridus fused to the regulatory sequences of a nucleargene, and Wang et al. (Plant Biotech. J., 3:475, 2005), describingtransgenic alfalfa, Arabidopsis, and tobacco plants expressing the atzAgene from Pseudomonas sp. that were able to detoxify atrazine.

Bayley et al. (Theor. Appl. Genet., 83:645, 1992) describe the creationof 2,4-D-resistant transgenic tobacco and cotton plants using the 2,4-Dmonooxygenase gene tfdA from Alcaligenes eutrophus plasmid pJP5. U.S.Patent Application Publication No. 20030135879 describes the isolationof a gene for dicamba monooxygenase (DMO) from Psueodmonas maltophiliathat is involved in the conversion of dicamba to a non-toxic3,6-dichlorosalicylic acid and thus may be used for producing plantstolerant to this herbicide.

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

B. Disease and Pest Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with a cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.(Science, 266:789-793, 1994) (cloning of the tomato Cf-9 gene forresistance to Cladosporium flavum); Martin et al. (Science,262:1432-1436, 1993) (tomato Pto gene for resistance to Pseudomonassyringae pv. tomato); and Mindrinos et al. (Cell, 78(6):1089-1099, 1994)(Arabidopsis RPS2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived and related viruses. See Beachyet al. (Ann. Rev. Phytopathol., 28:451, 1990). Coat protein-mediatedresistance has been conferred upon transformed plants against alfalfamosaic virus, cucumber mosaic virus, tobacco streak virus, potato virusX, potato virus Y, tobacco etch virus, tobacco rattle virus, and tobaccomosaic virus.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al. (Nature, 366:469-472, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Virus resistance has also been described in, for example, U.S.Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023 and5,304,730. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example genes homologous to theArabidopsis thaliana NIM1/NPR1/SAI1, and/or by increasing salicylic acidproduction (Ryals et al., Plant Cell, 8:1809-1819, 1996).

Logemann et al. (Biotechnology, 10:305-308, 1992), for example, disclosetransgenic plants expressing a barley ribosome-inactivating gene thathave an increased resistance to fungal disease. Plant defensins may beused to provide resistance to fungal pathogens (Thomma et al., Planta,216:193-202, 2002). Other examples of fungal disease resistance areprovided in U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407;6,215,048; 5,516,671; 5,773,696; 6,121,436; and 6,316,407.

Nematode resistance has been described in, for example, U.S. Pat. No.6,228,992, and bacterial disease resistance has been described in, forexample, U.S. Pat. No. 5,516,671.

The use of the herbicide glyphosate for disease control in soybeanplants containing event MON89788, which confers glyphosate tolerance,has also been described in U.S. Pat. No. 7,608,761.

C. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof, or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al. (Gene, 48(1):109-118,1986), who disclose the cloning and nucleotide sequence of a Bacillusthuringiensis δ-endotoxin gene. Moreover, DNA molecules encodingδ-endotoxin genes can be purchased from the American Type CultureCollection, Manassas, Va., for example, under ATCC Accession Nos. 40098,67136, 31995 and 31998. Another example is a lectin. See, for example,Van Damme et al., (Plant Molec. Biol., 24:825-830, 1994), who disclosethe nucleotide sequences of several Clivia miniata mannose-bindinglectin genes. A vitamin-binding protein may also be used, such as, forexample, avidin. See PCT Application No. US93/06487, the contents ofwhich are hereby incorporated by reference. This application teaches theuse of avidin and avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,protease, proteinase, or amylase inhibitors. See, for example, Abe etal. (J. Biol. Chem., 262:16793-16797, 1987) describing the nucleotidesequence of a rice cysteine proteinase inhibitor; Linthorst et al.(Plant Molec. Biol., 21:985-992, 1993) describing the nucleotidesequence of a cDNA encoding tobacco proteinase inhibitor I; and Sumitaniet al. (Biosci. Biotech. Biochem., 57:1243-1248, 1993) describing thenucleotide sequence of a Streptomyces nitrosporeus α-amylase inhibitor.

An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al. (Nature, 344:458-461, 1990) ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone; Gade and Goldsworthy (Eds.Physiological System in Insects, Elsevier Academic Press, Burlington,Mass., 2007), describing allostatins and their potential use in pestcontrol; and Palli et al. (Vitam. Horm., 73:59-100, 2005), disclosinguse of ecdysteroid and ecdysteroid receptor in agriculture. The diuretichormone receptor (DHR) was identified in Price et al. (Insect Mol.Biol., 13:469-480, 2004) as another potential candidate target ofinsecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al. (Seventh Int'l Symposium on Molecular Plant-MicrobeInteractions, Edinburgh, Scotland, Abstract W97, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments. Numerous other examples of insectresistance have been described. See, for example, U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245 and 5,763,241.

D. Male Sterility

Genetic male sterility is available in soybeans and, although notrequired for crossing soybean plants, can increase the efficiency withwhich hybrids are made, as it eliminates the need to physicallyemasculate the soybean plant used as a female in a given cross. (Brimand Stuber, Crop Sci., 13:528-530, 1973). Herbicide-inducible malesterility systems have also been described in, for example, U.S. Pat.No. 6,762,344.

When one desires to employ male-sterility systems, it may be beneficialto also utilize one or more male-fertility restorer genes. For example,when cytoplasmic male sterility (CMS) is used, hybrid seed productionrequires three inbred lines: (1) a cytoplasmically male-sterile linehaving a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm,which is isogenic with the CMS line for nuclear genes (“maintainerline”); and (3) a distinct, fertile inbred with normal cytoplasm,carrying a fertility restoring gene (“restorer” line). The CMS line ispropagated by pollination with the maintainer line, and all of theprogeny are male sterile, as the CMS cytoplasm is derived from thefemale parent. These male sterile plants can then be efficientlyemployed as the female parent in hybrid crosses with the restorer line,without the need for physical emasculation of the male reproductiveparts of the female parent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the soybean plant is utilized, butin many cases the seeds will be deemed the most valuable portion of thecrop, so fertility of the hybrids in these crops must be restored.Therefore, one aspect of the current invention concerns plants of thesoybean variety 01064164 comprising a genetic locus capable of restoringmale fertility in an otherwise male-sterile plant. Examples ofmale-sterility genes and corresponding restorers which could be employedwith the plants of the invention are well known to those of skill in theart of plant breeding, see, for example, U.S. Pat. Nos. 5,530,191 and5,684,242, the disclosures of which are each specifically incorporatedherein by reference in their entirety.

E. Modified Fatty Acid, Phytate, and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used, see Knutzon et al.(Proc. Natl. Acad. Sci. USA, 89:2624-2628, 1992). Various fatty aciddesaturases have also been described. McDonough et al. describe aSaccharomyces cerevisiae OLE1 gene encoding Δ9-fatty acid desaturase, anenzyme which forms the monounsaturated palmitoleic (16:1) and oleic(18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA (J.Biol. Chem., 267(9):5931-5936, 1992). Fox et al. describe a geneencoding a stearoyl-acyl carrier protein delta-9 desaturase from castor(Proc. Natl. Acad. Sci. USA, 90(6):2486-2490, 1993). Reddy et al.describe Δ6- and Δ12-desaturases from the cyanobacteria Synechocystisresponsible for the conversion of linoleic acid (18:2) togamma-linolenic acid (18:3 gamma) (Plant Mol. Biol., 22(2):293-300,1993). Arondel et al. describe a gene from Arabidopsis thaliana thatencodes an omega-3 desaturase has been identified (Science,258(5086):1353-1355, 1992). Plant Δ9-desaturases as well as soybean andBrassica Δ15-desaturases have also been described, see PCT ApplicationPublication No. WO 91/13972 and European Patent Application PublicationNo. EP0616644, respectively. U.S. Pat. No. 7,622,632 describes fungalΔ15-desaturases and their use in plants. European Patent ApplicationPublication No. EP1656449 describes Δ6-desaturases from Primula as wellas soybean plants having increased stearidonic acid (SDA, 18:4) content.U.S. Pat. No. 8,378,186 describes expression of transgenic desaturaseenzymes in corn plants, and improved fatty acid profiles resultingtherefrom.

Modified oil production is disclosed in, for example, U.S. Pat. Nos.6,444,876; 6,426,447; and 6,380,462. High oil production is disclosedin, for example, U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and6,476,295. Modified fatty acid content is disclosed in, for example,U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849;6,596,538; 6,589,767; 6,537,750; 6,489,461 and 6,459,018.

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al. (Gene, 127:87-94, 1993), for a disclosure of the nucleotidesequence of an Aspergillus niger phytase gene. For example, this couldbe accomplished in soybean plants by cloning and then reintroducing DNAassociated with the single allele that is responsible for soybeanmutants characterized by low levels of phytic acid. See Raboy et al.(Plant Physiol., 124(1):355-368, 2000).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. For example,Shiroza et al. (J. Bacteriol., 170:810-816, 1988) describe a nucleotidesequence of the Streptococcus mutans fructosyltransferase gene;Steinmetz et al. (Mol. Gen. Genet., 20:220-228, 1985) describe anucleotide sequence of the Bacillus subtilis levansucrase gene; Pen etal. (Biotechnology, 10:292-296, 1992) describe production of transgenicplants that express Bacillus licheniformis α-amylase; Elliot et al.(Plant Molec. Biol., 21:515-524, 1993) describe nucleotide sequences oftomato invertase genes; Sergaard et al. (J. Biol. Chem., 268:22480,1993) describe site-directed mutagenesis of a barley α-amylase gene; andFisher et al. (Plant Physiol., 102:1045-1046, 1993) describe maizeendosperm starch branching enzyme II. The Z10 gene encoding a 10 kD zeinstorage protein from maize may also be used to alter the quantities of10 kD zein in the cells relative to other components (Kirihara et al.,Gene, 71(2):359-370, 1988).

F. Resistance to Abiotic Stress

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, see U.S. Pat. No. 5,538,878.

G. Additional Traits

Additional traits can be introduced into the soybean variety of thepresent invention. A non-limiting example of such a trait is a codingsequence which decreases RNA and/or protein levels. The decreased RNAand/or protein levels may be achieved through RNAi methods, such asthose described in U.S. Pat. No. 6,506,559.

Another trait that may find use with the soybean variety of theinvention is a sequence which allows for site-specific recombination.Examples of such sequences include the FRT sequence used with the FLPrecombinase (Zhu and Sadowski, J. Biol. Chem., 270:23044-23054, 1995)and the LOX sequence used with CRE recombinase (Sauer, Mol. Cell. Biol.,7:2087-2096, 1987). The recombinase genes can be encoded at any locationwithin the genome of the soybean plant and are active in the hemizygousstate.

In certain embodiments soybean plants may be made more tolerant to ormore easily transformed with Agrobacterium tumefaciens. For example,expression of p53 and iap, two baculovirus cell-death suppressor genes,inhibited tissue necrosis and DNA cleavage. Additional targets mayinclude plant-encoded proteins that interact with the Agrobacterium Virgenes; enzymes involved in plant cell wall formation; and histones,histone acetyltransferases and histone deacetylases (reviewed in Gelvin,Microbiology & Mol. Biol. Reviews, 67:16-37, 2003).

In addition to the modification of oil, fatty acid, or phytate contentdescribed above, certain embodiments may modify the amounts or levels ofother compounds. For example, the amount or composition of antioxidantscan be altered. See, for example, U.S. Pat. Nos. 6,787,618 and 7,154,029and International Patent Application Publication No. WO 00/68393, whichdisclose the manipulation of antioxidant levels, and InternationalPatent Application Publication No. WO 03/082899, which discloses themanipulation of an antioxidant biosynthetic pathway.

Additionally, seed amino acid content may be manipulated. U.S. Pat. No.5,850,016 and International Patent Application Publication No. WO99/40209 disclose the alteration of the amino acid compositions ofseeds. U.S. Pat. Nos. 6,080,913 and 6,127,600 disclose methods ofincreasing accumulation of essential amino acids in seeds.

U.S. Pat. No. 5,559,223 describes synthetic storage proteins of whichthe levels of essential amino acids can be manipulated. InternationalPatent Application Publication No. WO 99/29882 discloses methods foraltering amino acid content of proteins. International PatentApplication Publication No. WO 98/20133 describes proteins with enhancedlevels of essential amino acids. International Patent ApplicationPublication No. WO 98/56935 and U.S. Pat. Nos. 6,346,403; 6,441,274; and6,664,445 disclose plant amino acid biosynthetic enzymes. InternationalPatent Application Publication No. WO 98/45458 describes synthetic seedproteins having a higher percentage of essential amino acids thanwild-type.

U.S. Pat. No. 5,633,436 discloses plants comprising a higher content ofsulfur-containing amino acids; U.S. Pat. No. 5,885,801 discloses plantscomprising a high threonine content; U.S. Pat. Nos. 5,885,802 and5,912,414 disclose plants comprising a high methionine content; U.S.Pat. No. 5,990,389 discloses plants comprising a high lysine content;U.S. Pat. No. 6,459,019 discloses plants comprising an increased lysineand threonine content; International Patent Application Publication No.WO 98/42831 discloses plants comprising a high lysine content;International Patent Application Publication No. WO 96/01905 disclosesplants comprising a high threonine content; and International PatentApplication Publication No. WO 95/15392 discloses plants comprising ahigh lysine content.

Origin and Breeding History of an Exemplary Single Locus Converted Plant

It is known to those of skill in the art that, by way of the techniqueof backcrossing, one or more traits may be introduced into a givenvariety while otherwise retaining essentially all of the traits of thatvariety. An example of such backcrossing to introduce a trait into astarting variety is described in U.S. Pat. No. 6,140,556, the entiredisclosure of which is specifically incorporated herein by reference.The procedure described in U.S. Pat. No. 6,140,556 can be summarized asfollows: The soybean variety known as Williams '82 [Glycine max L.Merr.] (Reg. No. 222, PI 518671) was developed using backcrossingtechniques to transfer a locus comprising the Rps₁ gene to the varietyWilliams (Bernard and Cremeens, Crop Sci., 28:1027-1028, 1988). Williams'82 is a composite of four resistant lines from the BC₆F₃ generation,which were selected from 12 field-tested resistant lines fromWilliams×Kingwa. The variety Williams was used as the recurrent parentin the backcross and the variety Kingwa was used as the source of theRps₁ locus. This gene locus confers resistance to 19 of the 24 races ofthe fungal agent Phytophthora root rot.

The F₁ or F₂ seedlings from each backcross round were tested forresistance to the fungus by hypocotyl inoculation using the inoculum ofrace 5. The final generation was tested using inoculum of races 1 to 9.In a backcross such as this, in which the desired characteristic beingtransferred to the recurrent parent is controlled by a major gene whichcan be readily evaluated during the backcrossing, it is common toconduct enough backcrosses to avoid testing individual progeny forspecific traits such as yield in extensive replicated tests. In general,four or more backcrosses are used when there is no evaluation of theprogeny for specific traits. As in this example, lines with thephenotype of the recurrent parent may be composited without the usualreplicated tests for traits, such as yield, protein, or oil percentage,in the individual lines.

The variety Williams '82 is comparable to the recurrent parent varietyWilliams in its traits except resistance to Phytophthora rot. Forexample, both varieties have a relative maturity of 38, indeterminatestems, white flowers, brown pubescence, tan pods at maturity, and shinyyellow seeds with black to light black hila.

Tissue Cultures and In Vitro Regeneration of Soybean Plants

A further aspect of the invention relates to tissue cultures of thesoybean variety designated 01064164. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calli,and plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In one embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves, or anthers.

Exemplary procedures for preparing tissue cultures of regenerablesoybean cells and regenerating soybean plants therefrom are disclosed inU.S. Pat. Nos. 4,992,375; 5,015,580; 5,024,944; and 5,416,011, each ofwhich are specifically incorporated herein by reference in theirentirety.

An important ability of a tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Soybeans typically are regenerated via two distinct processes: shootmorphogenesis and somatic embryogenesis (Finer, Cheng, Verma, “Soybeantransformation: Technologies and progress,” In: Soybean: Genetics,Molecular Biology and Biotechnology, CAB Intl, Verma and Shoemaker (ed),Wallingford, Oxon, UK, 250-251, 1996). Shoot morphogenesis is theprocess of shoot meristem organization and development. Shoots grow outfrom a source tissue and are excised and rooted to obtain an intactplant. During somatic embryogenesis, an embryo (similar to the zygoticembryo), containing both shoot and root axes, is formed from somaticplant tissue. An intact plant rather than a rooted shoot results fromthe germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show variety-specific responsesin which some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an ‘induction’ step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each soybean line. These optimizations mayreadily be carried out by one of skill in the art of tissue culturethrough small-scale culture studies. In addition to line-specificresponses, proliferative cultures can be observed with both shootmorphogenesis and somatic embryogenesis. Proliferation is beneficial forboth systems as it allows a single, transformed cell to multiply to thepoint that it will contribute to germ-line tissue.

Shoot morphogenesis was first reported by Wright et al. (Plant CellReports, 5:150-154, 1986) as a system from which shoots were obtained denovo from cotyledonary nodes of soybean seedlings. The shoot meristemswere formed subepidermally and morphogenic tissue could proliferate on amedium containing benzyl adenine (BA). This system can be used fortransformation if the subepidermal, multicellular origin of the shootsis recognized and proliferative cultures are utilized. The idea is totarget tissue that will give rise to new shoots and proliferate thosecells within the meristematic tissue to lessen problems associated withchimerism. Formation of chimeras, as a result of transforming only asingle cell in a meristem, is problematic if the transformed cell is notadequately proliferated and does not does not give rise to germ-linetissue. Once the system is well understood and reproducedsatisfactorily, it can be used as one target tissue for soybeantransformation.

Somatic embryogenesis in soybean was first reported by Christianson etal. (Science, 222:632-634, 1983) as a system in which embryogenic tissuewas initially obtained from the zygotic embryo axis. These embryogeniccultures were proliferative but the repeatability of the system was lowand the origin of the embryos was not reported. Later histologicalstudies of a different proliferative embryogenic soybean culture showedthat proliferative embryos were of apical or surface origin with a smallnumber of cells contributing to embryo formation. The origin of primaryembryos, the first embryos derived from the initial explant, isdependent on the explant tissue and the auxin levels in the inductionmedium (Hartweck et al., In Vitro Cell. Develop. Bio., 24:821-828,1988). With proliferative embryonic cultures, single cells or smallgroups of surface cells of the ‘older’ somatic embryos form the ‘newer’embryos.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

Definitions

In the description and tables, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

About: Refers to embodiments or values that include the standarddeviation of the mean for a given item being measured.

Allele: Any of one or more alternative forms of a locus. In a diploidcell or organism, the two alleles of a given locus occupy correspondingloci on a pair of homologous chromosomes.

Aphids: Aphid resistance in greenhouse screening is scored based onfoliar symptoms and number of aphids using a 1 to 9 scale. “Resistant”(R) corresponds to a rating between “1” and “3.9,” inclusive.“Moderately Resistant” (MR) corresponds to a rating between “4.0” and“5.9,” inclusive. “Moderately Susceptible to Moderately Resistant”(MS-MR) corresponds to a rating between “6.0” and “6.9,” inclusive.“Susceptible” (S) corresponds to a rating between “7.0” and “9.0,”inclusive.

Asian Soybean Rust (ASR): ASR may be visually scored based on a 1 to 5scale. A score of “1” indicates “immune.” A score of “2” indicates thatthe leaves exhibit red/brown lesions over less than 50% of surface. Ascore of “3” indicates that the leaves exhibit red/brown lesions overgreater than 50% of surface. A score of “4” indicates that the leavesexhibit tan lesions over less than 50% of surface. A score of “5”indicates that the leaves exhibit tan lesions over greater than 50% ofsurface. Resistance to ASR may be characterized phenotypically as wellas genetically. Soybean plants phenotypically characterized as resistantto ASR typically exhibit red/brown lesions covering less than 25% of theleaf. Genetic characterization of ASR resistance may be carried out, forexample, by identifying the presence in a soybean plant of one or moregenetic markers linked to the ASR resistance.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Brown Stem Rot (BSR): The greenhouse score is based on the incidence andseverity of pith discoloration and scores are converted to a 1 to 9scale. “Resistant” (R) corresponds to a rating less than “3.1.”“Moderately Resistant” (MR) corresponds to a rating between “3.1” and“5.0,” inclusive. “Moderately Susceptible” (MS) corresponds to a ratingbetween “5.1” and “6.9,” inclusive. “Susceptible” (S) corresponds to arating between “7.0” and “7.9,” inclusive. “Highly Susceptible” (HS)corresponds to a rating greater than “7.9.”

Chloride Sensitivity: Plants may be categorized as “includers” or“excluders” with respect to chloride sensitivity. Excluders tend topartition chloride in the root systems and reduce the amount of chloridetransported to more sensitive, aboveground tissues. Therefore excludersmay display increased tolerance to elevated soil chloride levels whencompared against includers. Greenhouse screening of chloride toleranceis scored on a 1 to 9 scale. A rating less than “3” is considered anexcluder. A rating between “4” and “9,” inclusive, is considered anincluder.

Chromatography: A technique in which a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Emergence (EMR): EMR describes the ability of a seed to emerge from thesoil after planting. Each genotype is scored based on its percent ofemergence using a 1 to 9 scale. Scoring a “1” indicates an excellentrate and percent of emergence. Scoring an intermediate score, such as“5,” indicates an average rate and percent of emergence. Scoring a “9”indicates a very poor rate and percent of emergence.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Fatty Acids: Are measured and reported as a percent of the total oilcontent. In addition to the typical composition of fatty acids incommodity soybeans, some soybean varieties have modified profiles. Lowlinolenic acid soybean oil as defined herein contains 3% or lesslinolenic acid. Mid oleic acid soybean oil as defined herein typicallycontains 50-60% oleic acid. High oleic soybean oil as defined hereintypically contains 75% or greater oleic acid. Stearidonic acid levelsare typically 0% in commodity soybeans.

Frog Eye Leaf Spot (FELS): Greenhouse assay reaction scores are based onfoliar symptom severity and are measured using a 1-9 scale. “Resistant”(R) corresponds to a rating less than “3.” “Moderately Resistant” (MR)corresponds to a rating between “3.0” and “4.9,” inclusive. “ModeratelySusceptible” (MS) corresponds to a rating between “5.0” and “6.9,”inclusive. “Susceptible” (S) corresponds to a rating greater than 6.9.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Iron-Deficiency Chlorosis (IDE=early; IDL=late): Iron-deficiencychlorosis is scored based on visual observations using 1 to 9 scale. Ascore of “1” indicates that no stunting of the plants or yellowing ofthe leaves was observed. A score of “9” indicates that the plants aredead or dying as a result of iron-deficiency chlorosis. A score of “5”means that plants display intermediate health and some observable leafyellowing.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Linolenic Acid Content (LLN): Low-linolenic acid soybean oil contains 3%or less linolenic acid. Traditional soybean oil contains approximately8% linolenic acid.

Lodging Resistance (LDG): LDG is rated on a scale of 1 to 9. A score of“1” indicates that the plants are erect. A score of “5” indicates thatthe plants are leaning at a 45 degree angle in relation to the ground. Ascore of “9” indicates that the plants are lying on the ground.

Marker: A readily detectable phenotype, preferably inherited inco-dominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Maturity Date (MAT): Plants are considered mature when 95% of the podshave reached their mature color. The maturity date is typicallydescribed in measured days after August 31 in the northern hemisphere.

Moisture (MST): The average percentage moisture in the seeds of avariety.

Oil or Oil Percent: Seed oil content is measured and reported on apercentage basis.

Or: As used herein is meant to mean “and/or” and be interchangeabletherewith unless explicitly indicated to refer to the alternative only.

Phenotype: The detectable characteristics of a cell or organism, whichare the manifestation of gene expression.

Phenotypic Score (PSC): The phenotypic score is a visual rating of thegeneral appearance of the variety. All visual traits are considered inthe score, including healthiness, standability, appearance, and freedomfrom disease. Ratings are scored as 1 being poor to 9 being excellent.

Phytophthora Allele: Susceptibility or resistance to Phytophthora rootrot races is affected by alleles such as Rps1a (denotes resistance toRaces 1, 2, 10, 11, 13-18, 24, 26, 27, 31, 32, and 36); Rps1c (denotesresistance to Races 1-3, 6-11, 13, 15, 17, 21, 23, 24, 26, 28-30, 32, 34and 36); Rps1k (denotes resistance to Races 1-11, 13-15, 17, 18, 21-24,26, 36 and 37); Rps2 (denotes resistance to Races 1-5, 9-29, 33, 34 and36-39); Rps3a (denotes resistance to Races 1-5, 8, 9, 11, 13, 14, 16,18, 23, 25, 28, 29, 31-35); Rps6 (denotes resistance to Races 1-4, 10,12, 14-16, 18-21 and 25); and Rps7 (denotes resistance to Races 2, 12,16, 18, 19, 33, 35 and 36).

Phytophthora Root Rot (PRR): Disorder of which the most recognizablesymptom is stem rot. Brown discoloration ranges below the soil line andup to several inches above the soil line. The leaves often turn yellow,dull green and/or gray and may become brown and wilted, but remainattached to the plant.

Phytophthora Tolerance: Tolerance to Phytophthora root rot is rated inthe greenhouse assay based on a 1 to 9 scale. A rating less than “3.5”indicates “tolerant.” A rating between “3.5” and “6,” inclusive,indicates “moderately tolerant.” A rating greater than “6” indicates“sensitive.” Note that a score between “1” and “2” may indicateresistance to Phytophthora and therefore not be a true reflection ofhigh tolerance to Phytophthora.

Plant Height (PHT): Plant height is taken from the top of soil to thetop node of the plant and is measured in inches.

Predicted Relative Maturity (PRM): The maturity grouping designated bythe soybean industry over a given growing area. This figure is generallydivided into tenths of a relative maturity group. Within narrowcomparisons, the difference of a tenth of a relative maturity groupequates very roughly to a day difference in maturity at harvest.

Protein (PRO), or Protein Percent: Seed protein content is measured andreported on a percentage basis.

Regeneration: The development of a plant from tissue culture.

Relative Maturity: The maturity grouping designated by the soybeanindustry over a given growing area. This figure is generally dividedinto tenths of a relative maturity group. Within narrow comparisons, thedifference of a tenth of a relative maturity group equates very roughlyto a day difference in maturity at harvest.

Seed Protein Peroxidase Activity: Seed protein peroxidase activity isdefined as a chemical taxonomic technique to separate varieties based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean varieties, those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Weight (SWT): Soybean seeds vary in size; therefore, the number ofseeds required to make up one pound also varies. This affects the poundsof seed required to plant a given area and can also impact end uses.“SW100” equals the weight in grams of 100 seeds.

Seed Yield (Bushels/Acre): The yield in bushels/acre is the actual yieldof the grain at harvest.

Seedling Vigor Rating (SDV): General health of the seedling that ismeasured on a 1 to 9 scale in which “1” is “best” and “9” is “worst.”

Seeds per Pound: Soybean seeds vary in size; therefore, the number ofseeds required to make up one pound also varies. This affects the poundsof seed required to plant a given area and can also impact end uses.

Selection Index (SELIN): The percentage of the test mean.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Shattering: The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of “1” indicates that the pods have not opened and no seeds havefallen out. A score of “5” indicates that approximately 50% of the podshave opened, with seeds falling to the ground. A score of “9” indicatesthat 100% of the pods are opened.

Single Locus Converted (Conversion) Plant: Plants that are developed bya plant breeding technique called backcrossing and/or by genetictransformation to introduce a given locus that is transgenic in origin,in which essentially all of the morphological and physiologicalcharacteristics of a soybean variety are recovered in addition to thecharacteristics of the locus transferred into the variety via thebackcrossing technique or by genetic transformation. It is understoodthat once introduced into any soybean plant genome, a locus that istransgenic in origin (transgene), can be introduced by backcrossing aswith any other locus.

Southern Root Knot Nematode (SRKN): Greenhouse assay reaction scores arebased on severity and measured using a 1 to 9 scale. “Resistant” (R)corresponds to a rating less than “6.1.” “Moderately Resistant” (MR)corresponds to a rating between “6.1” and “6.6,” inclusive. “ModeratelyResistant to Moderately Susceptible” (MR-MS) corresponds to a ratingbetween “6.6” and “7.4,” inclusive. “Susceptible” (S) corresponds to arating great than “7.4.”

Southern Stem Canker (STC): Greenhouse assay scoring is based onpercentage of dead plants (DP). This percentage is converted to a 1 to 9scale: “1” corresponds to no DP. “2” corresponds to less than 10% DP.“3” corresponds to between 10% and 30%, inclusive, DP. “4” correspondsto between 31% and 40%, inclusive, DP. “5” corresponds to between 41%and 50%, inclusive, DP. “6” corresponds to between 51% and 60%,inclusive, DP. “7” corresponds to between 61%-70%, inclusive, DP. “8”corresponds to between 71% and 90%, inclusive, DP. “9” corresponds tobetween 91% and 100%, inclusive, DP. “Resistant” (R) corresponds to arating less than “3.9.” “Moderately Resistant” (MR) corresponds to arating between “4” and “5.9,” inclusive. “Moderately Susceptible” (MS)corresponds to a rating between “6” and “7.9,” inclusive. “Susceptible”(S) corresponds to a rating between “8” and “8.9,” inclusive. “HighlySusceptible” (HS) corresponds to a rating greater than “8.9.”

Soybean Cyst Nematode (SCN): Greenhouse screening scores are based on afemale index % of Lee 74. “Resistant” (R) corresponds to a rating lessthan 10%. “Moderately Resistant” (MR) corresponds to a rating between10% and 21.9%, inclusive. “Moderately Resistant to ModeratelySusceptible” (MR-MS) corresponds to a rating between 22% and 39.9%,inclusive. “Susceptible” (S) corresponds to a rating greater than 39.9%.

Stearate: A fatty acid in soybean seeds measured and reported as apercent of the total oil content.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference from the mean, e.g., p=0.05.

Sudden Death Syndrome: Leaf symptoms first appear as bright yellowchlorotic spots with progressive development of brown necrotic areas andeventual leaflet drop. Greenhouse screening plants are scored based onfoliar symptom severity using a 1 to 9 scale. “Resistant” (R)corresponds to a rating less than “3.” “Moderately Resistant” (MR)corresponds to a rating between “3.0” and “4.9,” inclusive. “ModeratelySusceptible” (MS) corresponds to a rating between “5.0” and “6.9,”inclusive. “Susceptible” (S) corresponds to a rating between “7.0” and“8.0,” inclusive. “Highly Susceptible” (HS) corresponds to a ratinggreater than “8.”

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a soybean plant by transformation orsite-specific recombination.

Yield Best Estimate (YLD_BE): The adjusted yield of a plot inbushels/acre. Plot yields are adjusted using the nearest neighborspatial covariate method first described by Papadakis (Méthodestatistique pour des experiences sur champ, Thessaloniki Plant BreedingInstitute Bulletin No. 23, Thessaloniki, London, 1937).

Yield Count (YLD_COUNT): The number of evaluated plots.

DEPOSIT INFORMATION

A deposit of the soybean variety 01064164, which is disclosed hereinabove and referenced in the claims, will be made with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209. The date of deposit is Feb. 26, 2018 and the accessionnumber for those deposited seeds of soybean variety 01064164 is ATCCAccession No. PTA-124954. All restrictions upon the deposit have beenremoved, and the deposit is intended to meet all of the requirements ofthe Budapest Treaty and 37 C.F.R. § 1.801-1.809. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed:
 1. A plant of soybean variety 01064164, whereinrepresentative seed of said soybean variety have been deposited underATCC Accession No. PTA-124954.
 2. A plant part of the plant of claim 1,wherein the plant part comprises at least one cell of said plant.
 3. Aseed of soybean variety 01064164, wherein representative seed of saidsoybean variety have been deposited under ATCC Accession No. PTA-124954.4. A method of producing soybean seed, the method comprising crossingthe plant of claim 1 with itself or a second soybean plant to producesaid soybean seed.
 5. The method of claim 4, the method furthercomprising crossing the plant of soybean variety 01064164 with a second,nonisogenic soybean plant to produce said soybean seed.
 6. An F₁ soybeanseed produced by the method of claim
 5. 7. A soybean plant produced bygrowing the F₁ soybean seed of claim
 6. 8. A composition comprising theseed of claim 3 comprised in plant seed growth media.
 9. The compositionof claim 8, wherein the plant seed growth media is soil or a syntheticcultivation medium.
 10. A plant of soybean variety 01064164 furthercomprising a single locus conversion, wherein said plant otherwisecomprises all of the morphological and physiological characteristics ofsaid soybean variety when grown under the same environmental conditions,and wherein representative seed of said soybean variety have beendeposited under ATCC Accession No. PTA-124954.
 11. The plant of claim10, wherein the single locus conversion comprises a transgene.
 12. Aseed that produces the plant of claim
 10. 13. The seed of claim 12,wherein the single locus confers a trait selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, modified fatty acid metabolism,abiotic stress resistance, altered seed amino acid composition,site-specific genetic recombination, and modified carbohydratemetabolism.
 14. The seed of claim 13, wherein the single locus thatconfers herbicide tolerance confers tolerance to benzonitrileherbicides, cyclohexanedione herbicides, imidazolinone herbicides,phenoxy herbicides, sulfonylurea herbicides, triazine herbicides,1-aminocyclopropane-1-carboxylic acid synthase-inhibiting herbicides,4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicides, acetolactatesynthase-inhibiting herbicides, protoporphyrinogen oxidase-inhibitingherbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), bromoxynil, dicamba,glufosinate, glyphosate, nicosulfuron, or quizalofop-p-ethyl.
 15. Theseed of claim 12, wherein the single locus conversion comprises atransgene.
 16. The method of claim 5, the method further comprising: (a)crossing a plant grown from said soybean seed with itself or a differentsoybean plant to produce seed of a progeny plant of a subsequentgeneration; (b) growing a progeny plant of a subsequent generation fromsaid seed of a progeny plant of a subsequent generation and crossing theprogeny plant of a subsequent generation with itself or a second plantto produce seed of a progeny plant of a further subsequent generation;and (c) repeating step (b) with sufficient inbreeding to produce seed ofan inbred soybean plant that is derived from soybean variety 01064164.17. The method of claim 16, the method further comprising crossing aplant grown from said seed of an inbred soybean plant that is derivedfrom soybean variety 01064164 with a nonisogenic plant to produce seedof a hybrid soybean plant that is derived from soybean variety 01064164.18. A method of producing a commodity plant product, the methodcomprising producing the commodity plant product from the plant ofclaim
 1. 19. The method of claim 18, wherein the commodity plant productis selected from a group comprising protein concentrate, proteinisolate, grain, soybean hulls, meal, flour, and oil.
 20. A commodityplant product that is produced by the method of claim 18, wherein thecommodity plant product comprises at least one cell of soybean variety01064164.